1. Field of the Invention
The invention relates to a transgenic cell line which has been engineered to constitutively express the αVβ6 integrin receptor, a principal bovine receptor for Foot and Mouth Disease Virus (FMDV). In particular, the invention relates to the transgenic fetal porcine kidney cell line, LFBK αvβ6, which is useful for the rapid and sensitive detection and identification of FMDV in diagnostic settings and also to identify serotypes and subtypes thereby facilitating vaccine selection procedures. LFBK αvβ6 is highly sensitive and permissive to infection by animal-derived FMDV from all seven serotypes in cell culture. The invention further relates to the transgenic LFBK αvβ6 cells for detection of FMDV from field samples.
2. Description of the Relevant Art
Foot and mouth disease virus (FMDV) is a severe economic concern for meat producing nations since the trade of animal products is prohibited from countries where the virus is confirmed. The rapid spread of the virus among susceptible animals results in severe morbidity and in some cases death, especially in young animals (Grubman and Baxt. 2004. Clin. Microbiol. Rev. 17: 465-493). Foot and mouth disease (FMD) is an extremely contagious viral disease of cloven-hoofed ungulates which include domestic animals (cattle, pigs, sheep, goats, and others) and a variety of wild animals. Infection or vaccination with one of the seven different serotypes does not confer cross-protection to other serotypes or even some subtypes of the same serotype. Vaccines for FMDV are widely used to prevent clinical disease, but since vaccines are serotype and subtype-specific, the virus(es) causing outbreaks must be isolated and serologically characterized for vaccine matching prior to selecting the appropriate vaccine antigen (reviewed in Rodriguez and Gay. 2011. Expert Rev. Vaccines 10:377-387).
Although molecular techniques such as PCR (polymerase chain reaction) coupled with genomic sequencing can be used in samples containing enough virus to rapidly identify the virus serotype and its relationship to other FMDV strains, appropriate vaccine prediction requires virus growth in cell culture to carry out neutralization tests using reference sera. Inefficient recovery of virus from animal samples can delay diagnosis and vaccine selection and thereby hamper rapid implementation of control measures; therefore, virus isolation protocols are designed for maximum sensitivity. Some primary cells, such as bovine thyroid (BTY) cells, are highly susceptible to a wide range of FMDV serotypes (Snowdon, W. A. 1966. Nature 210:1079-1080); however, they are difficult and costly to prepare and lose FMDV susceptibility after multiple passages (House and Yedloutschnig. 1982. Can. J. Comp. Med. 46:186-189). Primary lamb kidney (LK) cells are also very sensitive to FMDV. Unlike BTY cells, LK cells maintain their sensitivity to FMDV infection after cryopreservation; however, their sensitivity decreases after passage (House and House. 1989. Vet. Microbiol. 20:99-109). Immortalized cell lines (e.g. baby hamster kidney (BHK) fibroblasts and porcine kidney epithelial cells), while much easier to maintain, are in many cases less susceptible to specific animal-derived FMDV serotypes (Swaney, L. M. 1976. Amer. J. Vet. Res. 37:1319-1322; Ferris et al. 2006. Vet. Microbiol. 117:130-140; Ferris et al. 2002. Vet. Microbiol. 84:307-316; De Castro, M. P. 1964. Arch. Inst. Biol. San Paulo 31: 63-78).
Integrins of the αV subgroup have been demonstrated to be FMDV receptors by several laboratories including ours (Ruiz-Saenz et al. 2009. Intervirol. 52:201-212). Of the many αV integrins that have been shown to mediate FMDV attachment, the integrin αVβ6 has been shown to be one of the most efficient receptors for all FMDV serotypes (Jackson et al. 2000. J. Virol. 74:4949-4956; Ferris et al. 2005. J. Virological Methods 127:69-79) and high levels of αVβ6 expression are observed on epithelial cells at the sites of infection in cattle and swine (Monaghan et al. 2005. J. Gen. Virol. 86:2769-2780; O'Donnell et al. 2009. J. Comp. Path. 141:98-112). BTY cells, considered the most sensitive primary cells for FMDV isolation, have high levels of αVβ6 integrin surface expression (King et al. 2011. Vet. Immunol. Immunopath. 140:259-265). Moreover, transient expression of bovine αV and β6 integrin subunits in baby hamster kidney cells (BHK3-αVβ6) (Duque et al. 2004. J. Virol. 78:9773-9781) greatly increased the susceptibility of this cell line to a cow-passaged A24 Cruziero strain that contains an SGD motif in the VP1 (FMD Virus Protein 1) capsid protein (Rieder et al. 2005. J. Virol. 79:12989-12998). Although the BHK3-αVβ6 cells were initially more permissive to the A24-SGD virus than BHK-21 cells were, the BHK3-αVβ6 cells lost integrin expression and sensitivity to the A24-SGD virus after multiple passages (E. Rieder, personal communication).
Swaney derived an immortalized line of fetal porcine kidney (LFBK) cells that had high susceptibility to most FMDV serotypes and the susceptibility was maintained over many passages (Swaney, L. M. 1988. Vet. Microbiol. 18:1-14). Compared to BTY cells, LFBK cells had similar susceptibility to most FMDV serotypes and had equal or better susceptibility than the MVPK (Mengeling-Vaughn Porcine Kidney) cell line, the porcine kidney cell line, IB-RS-2, and fetal bovine kidney cells in the same experiments.
There is a need for a cell line that is easily maintained and is highly susceptible to all serotypes and subtypes of FMDV. The present invention, described below, combines the long-lived FMDV susceptibility of the LFBK cell line with a principal bovine receptor for FMDV, the αVβ6 integrin receptor, to provide a stable cell line which is highly susceptible to FMDV.